Species differences in the behavioral effects of cerulein--an agonist of the receptors of the octapeptide cholecystokinin--in white mice and rats

It has been shown in the behavioural experiments that combined pretreatment with haloperidol (0.25 mg/kg) and caerulein (40 micrograms/kg), and to a lesser extent pretreatment with caerulein alone caused long-term reversal of amphetamine (2 mg/kg) induced hyperexcitability in rats. Administration of proglumide (50 mg/kg), an antagonist of CCK-8 receptors, did not reverse long-term antiamphetamine effect of caerulein. In mice pretreatment with caerulein (50 and 100 micrograms/kg) alone or in combination with haloperidol (0.25 mg/kg) caused hypersensitivity to the behavioural effect of amphetamine (3 mg/kg). Intraventricular (I ng), but not systemic (100-500 micrograms/kg) administration of caerulein selectively antagonized seizures in mice induced by intraventricular administration of quinolinic acid (5 micrograms) and N-methyl-D-aspartate (0.2 microgram). Pretreatment with proglumide (50 mg/kg) reversed the anticonvulsive effect of caerulein in mice. In rats, caerulein failed to affect the seizures caused by intraventricular administration of quinolinic acid. The results of the present study demonstrate the existence of obvious interspecies differences in the behavioural effects of caerulein, the agonist of CCK-8 receptors, in mice and rats.     

Enhanced binding of radioligands to receptors of γ-aminobutyric acid and benzodiazepine by a new anticonvulsive agent,LY81067

A diaryltriazine, LY81067, effectively protects against pentylenetetrazole- and picrotoxin-induced convulsions in mice, with ED₅₀ values of 5.7 and 5.8 mg/kg i.p., respectively. LY81067 enhances the binding of both ³H-GABA and ³H-flunitrazepam to specific sites in rat brain membranes. The degree of enhancement by LY81067 varies from one brain region to another and is different for the binding of ³H-GABA and ³H-flunitrazepam. In cortical membranes, LY81067 increases the affinity of ³H-GABA for both high and low affinity sites and increases the number of sites. LY81067 increases the affinity of ³H-flunitrazepam for its binding sites without greatly increasing the number of sites. Like the pyrazolopyridines, the enhancement of ³H-flunitrazepam binding by LY81067 is dependent on chloride or related anions and is reversed by picrotoxin, suggesting that LY81067 exerts its anticonvulsant effects by binding to or near picrotoxin binding sites. The differential effects of LY81067 on the enhancements of ³H-GABA and ³H-flunitrazepam binding in several brain regions suggest extensive multiplicity of GABA/benzodiazepine/picrotoxin/anioin receptor complexes.     

Effects of Picrotoxin Treatment on GABAA Receptor Supramolecular Complexes in Rat Brain

The effects of acute and chronic administration of a subconvulsive dose of picrotoxin on t-[35S]butylbicyclophosphorothionate ([3S]TBPS), [3H]muscimol, and [3H]flunitrazepam binding characteristics in various regions and on the convulsant potency of picrotoxin in Sprague-Dawley rats were examined. Acute administration of a subconvulsive dose of picrotoxin (3 mg/kg, i.p.) significantly increased [35S]TBPS and [3H]muscimol binding in cerebellum (CB) with no change in frontal cortex (FC). In rats treated chronically with picrotoxin (3 mg/kg, i.p., daily for 10 days), the Bmax of [35S]TBPS binding site was significantly decreased in the FC, striatum (ST), and CB with no change in KD values. Neither [3H]muscimol binding in the FC and CB nor [3H]flunitrazepam binding in the FC was affected in these rats. In addition, the potency of pentobarbital to inhibit [35S]TBPS binding in vitro was not altered following acute or chronic treatment of picrotoxin. Chronic administration of picrotoxin did not affect convulsive ED50 or LD50 of picrotoxin; however, it delayed the onset of convulsions and increased the time to death. These results suggest that treatment with picrotoxin at a subconvulsive dose for 10 days causes down-regulation of [35S]TBPS binding sites and that this down-regulation might be related, at least in part, to the decreased extent of convulsant potency of picrotoxin. In addition, the results indicate possible interaction between convulsant binding sites and GABAA receptor sites in the CB following picrotoxin treatment.     

Effect of treatment with submaximal and excessive doses of caerulein on pancreatic growth in newborn rats

Different groups of CFY female newborn rats were treated with saline, or 1 microgram/kg or 100 micrograms/kg doses of caerulein given s. c. 3 x/day. Application of 100 micrograms/kg dose of caerulein for 3 days stimulated pancreatic growth inducing pancreatic hyperplasia; both (1 and 100 micrograms/kg) doses evoked increase in trypsin/DNA ratio inducing pancreatic hypertrophy in 4-days-old rats. Using the indices as before application of 1 microgram/kg caerulein for 10 days stimulated pancreatic growth and both (1 and 100 micrograms/kg) doses elicited glandular hypertrophy in 11-days-old rats. In 24-old-rats the 1 microgram/kg doses of caerulein given for 3 days stimulated pancreatic growth and induced pancreatic hypertrophy, the 100 micrograms/kg doses of the peptide given for 3 days, however, evoked pancreatic aplasia and atrophy.     

Effect of lorglumide (CR-1409) on pancreatic secretory and trophic response to caerulein in newborn rats.

The authors investigated whether lorglumide a specific CCK-receptor antagonist affects the pancreatic actions of caerulein in female newborn Wistar rats. Pancreatic secretory response (expressed as the decrease in specific trypsin activity in the pancreas) was studied in 11-day-old rats following acute administration of saline (control), caerulein (0.3, 1, or 3 micrograms/kg s.c.) either without or with lorglumide (10 mg/kg s.c.). Lorglumide was given 15 min before caerulein. In chronic studies rats were treated 3x/day for 10 days from the day of birth (Day 1) with caerulein and lorglumide as above. On Day 11 the rats were decapitated and exsanguinated, their pancreas removed and analyzed. Acute administration of caerulein induced a dose-dependent depletion of specific trypsin activity from the pancreas and this was antagonized by lorglumide. Chronic treatment with each dose of the peptide increased total pancreatic trypsin content. Besides, the 3 micrograms/kg dose caused to increase pancreatic protein, DNA, and amylase content and to increase plasma corticosterone level. Chronic administration of lorglumide did not influence normal pancreatic growth, while it strongly inhibited the increase in trypsin content evoked by caerulein. However, lorglumide, given alone or in combination with caerulein, induced a significant increase in pancreatic amylase content without affecting plasma corticosterone level.     

Changes in the number of benzodiazepine receptors in different parts of the brain of rats after neuroleptic withdrawal

It has been demonstrated in experiments on rats receiving chronic (16 days) treatment with haloperidol (1.0 mg/kg/day), sulpiride (50 mg/kg/day) and clozapine (10 mg/kg/day) that binding of 3H-flunitrazepam in the striatum, limbic system, and cortex is reduced at the 5th day after withdrawal of the neuroleptics. That release was determined by the diminution of the number of receptors without changing in the dissociation constant. The reduction in the density of benzodiazepine receptors (BD-receptors) after withdrawal of the neuroleptics attests to their agonistic effect on BD-receptors. Apparently these changes are not linked with a direct effect of the neuroleptics on BD-receptors, since they displace 3H-flunitrazepam in experiments in vitro only at micromolar concentrations. It is assumed that the reduction in 3H-flunitrazepam binding is mediated via the GABAergic system transsynaptically in response to increase in the number of dopamine (neuroleptic) receptors.     

The participation of CCK-8-ergic mechanisms in the regulation of emotional behavior in rodents

Effects of CCK-8 receptor agonists caerulein and pentagastrin and CCK-8 receptor antagonist proglumide on exploratory and locomotor activity of mice and rats were studied. Systemic administration of caerulein (500 ng/kg 1 mcg/kg) decreased significantly the exploratory activity of mice in elevated plus-maze. This anxiogenic-like action of caerulein was attenuated by acute pretreatment with proglumide (1 and 15 mg/kg) but not with diazepam (up to 0.75 mg/kg). Proglumide slightly increased the exploratory activity of rats in plus-maze; on the other hand, caerulein and pentagastrin potently decreased the measures of exploration in this test. Caerulein (10-100 mcg/kg) and proglumide (1 and 15 mg/kg) inhibited 3H-pentagastrin binding in mice brain in in vivo experiments. The data obtained indicate that CCK-8-ergic mechanisms in brain play an important role in the generation of anxiety states in rodents.     

Neuroleptic-like properties of cholecystokinin analogs: distinctive mechanisms underlying similar behavioral profiles depending on the route of administration

Rats were trained to discriminate vehicle injections from intraperitoneal injections of 3 micrograms/kg caerulein, a cholecystokinin (CCK) neuropeptide analog. The reward that reinforced correct choices was an electrical brain stimulation self-administered by bar pressing. Dose-response quantitative generalization was obtained by using 1 and 2 micrograms/kg caerulein. Qualitative generalization to the vehicle occurred after injecting 10, 20 and 200 micrograms/kg unsulfated CCK-8, 10, 20 and 200 micrograms/kg CCK-4, 5 micrograms/kg CCK-8 and 1 microgram/kg caerulein, neurotensin or bombesin and 200 micrograms/kg apomorphine or 320 micrograms/kg amphetamine. Total generalization to the caerulein cue was obtained with 20 micrograms/kg sulfated CCK-8 or gastrin 2-17, 25 micrograms/kg somatostatin, 50 micrograms/kg haloperidol and 2 mg/kg chlorpromazine. The previous 5 mg/kg injection of an antiemetic drug such as chlorhydrate of trimethobenzamide did not eliminate the discriminative properties of a subsequent injection of caerulein. Our data thus tend to show that IP injection of caerulein produces effects similar to those of IP neuroleptics.     

Antagonism of caerulein, a CCK-8 receptor agonist, to the behavioral effects of ketamine in mice and rats

It has been established in experiments on male mice and rats that caerulein antagonized the behavioural effects of ketamine, an agonist of phencyclidine receptors. Caerulein (75-375 micrograms/kg) and haloperidol (0.1-1.5 mg/kg) suppressed the stereotyped behaviour and motor excitation induced by ketamine (30 mg/kg) in mice. Caerulein and haloperidol failed to affect ketamine-induced ataxia. Caerulein (10 micrograms/kg) and the opioid antagonist naloxone (5 mg/kg) completely blocked the amnestic action of ketamine (30 mg/kg) in passive avoidance experiments on rats. It seems likely that the suppression of the behavioural effects of ketamine by caerulein is related to its functional antagonism with dopamine and opioid receptors.     

Independent benzodiazepine and beta-carboline binding sites in the brain of aggressive and anxious-defensive mice

Distribution of specific 3H-flunitrazepam and 3H-beta-carboline-3-carboxylate binding sites in the brain regions of aggressive and timid-defensive mice was investigated before and after subchronic injection of diazepam (5 mg/kg). The absence of differences between the affinity and concentration of 3H-flunitrazepam binding sites in diencephalon and brain cortex in aggressive and defensive mice may be explained by general benzodiazepine receptor reaction on isolation and agonistic interaction stress. Significant predominance of 3H-beta-carboline-3-carboxylate binding sites in the brain cortex, as compared to the concentration of 3H-flunitrazepam binding sites suggests the presence of specific binding sites for beta-carbolines, which have specific distribution in the brain.     

Anticonvulsive and free radical scavenging activities of vanillyl alcohol in ferric chloride-induced epileptic seizures in Sprague-Dawley rats

Vanillyl alcohol (VA) is a component of Gastrodia elata Bl. (GE), which is a traditional Chinese herb widely used to treat convulsive disorders or dizziness. This study examined the role of VA in the anticonvulsive properties of GE in a Sprague-Dawley rat model of epilepsy. The anticonvulsive and free radical scavenging activities of VA were examined after intracortical injection of ferric chloride (100 mM, 8 microl) to induce epileptic seizures. These seizures were verified by behavioral observations and electroencephalographic (EEG) and electromyographic (EMG) recordings. Ferric chloride injection resulted in increased lipid peroxide levels in the ipsilateral and contralateral cerebral cortex, and increased luminol-chemiluminescence (CL) and lucigenin-CL counts in the peripheral blood. Intraperitoneal injection (i.p.) of VA (200 mg/kg or 100 mg/kg) or phenytoin 10 mg/kg prior to ferric chloride administration significantly inhibited wet dog shakes (WDS) and lipid peroxide levels in the bilateral cerebral cortex. VA 200 mg/kg also significantly reduced luminol-CL and lucigenin-CL counts in the peripheral blood, but no significant effect was observed following administration of VA 100 mg/kg or phenytoin. These data indicate that VA has both anticonvulsive and suppressive effects on seizures and lipid peroxidation induced by ferric chloride in rats. Data from the present study also demonstrate that VA has free radical scavenging activities, which may be responsible for its anticonvulsive propertics. This finding is consistent with the results from previous studies that generation of superoxide radical evoked by injection of iron salt into rat brain plays a critical role in ferric chloride-induced seizures. In addition, the results of the present study suggest that the anticonvulsive effect of GE may be attributable, at least in part, to its VA component.     

Effects of clonazepam on picrotoxin-induced convulsions.

The convulsant effects of four doses of picrotoxin (PX)-2, 3, 4, and 6 mg/kg s.c.-were evaluated in the first part of the study. The 4-mg/kg dose, which elicited minimal seizures in all animals, generalized tonic-clonic (major) seizures in 75% of rats and fatal outcome in 69% of rats, was chosen for the second part, i.e. for testing the anticonvulsant action of clonazepam (Rivotril Roche, 0.1 or 1 mg/kg i.p.). Clonazepam exhibited a dose-dependent action against PX-induced seizures, being more efficient against major than against minimal seizures.     

Modulation of benzodiazepine by lysine and pipecolic acid on pentylenetetrazol-induced seizures

L-lysine, an essential amino acid for man and animals, and its metabolite pipecolic acid (PA) have been studied for their effects on pentylenetetrazol (PTZ)-induced seizures in mice. L-Lysine or L-PA i.p. significantly increased clonic and tonic latencies in a dose-dependent manner against 90 mg/kg PTZ-induced seizures. L-Lysine but not L-PA enhanced the anticonvulsant effect of diazepam (DZ) (0.2 mg/kg). L-PA (0.1 mmol/kg) i.c.v. showed a slight decrease in clonic latency; it did not enhance the antiseizure activity of DZ; it caused seizures at 0.6 mmol/kg. D-PA (0.1 mmol/kg) i.c.v. displayed an opposite effect compared to its L-isomer. The anticonvulsant effect of L-lysine in terms of increase in seizure latency and survival was even more amplified when tested with a submaximal PTZ concentration (65 mg/kg). L-Lysine showed an enhancement of specific 3H-flunitrazepam (FZ) binding to mouse brain membranes both in vitro and in vivo. The possibility of L-lysine acting as a modulator for the GABA/benzodiazepine receptors was demonstrated. Since L-PA showed enhancement of 3H-FZ binding only in vitro but not in vivo, the anticonvulsant effect of L-PA may not be linked to the GABA/benzodiazepine receptor.     

Sex related differences in the response of mice, rats and cats to administration of picrotoxin

Picrotoxin, 2.5 mg/kg, which was subconvulsive in male rats was 92% convulsive in female rats. Four mg/kg of picrotoxin, a dose which did not produce death in the male rats, was 75% lethal in the female rats. Picrotoxin also produced a significantly greater increase in the frequency of the spinal motoneurons discharge in the female than in male rats (444% of control compared to 222% of control). A similar significant difference to the analogous treatment was obtained in the female and male cats (439% of control compared to 368% of control). To counteract the picrotoxin-induced increased frequency of the spinal motoneurons discharge a double dose of diazepam had to be given to females of both species. A sex related difference in the occurrence of convulsions, latency and death following picrotoxin administration was also present in mice. However, mice responded in an opposite direction to rats and cats. Three mg/kg of picrotoxin was 100% convulsive and 27% lethal in male mice, while only 40% convulsive and 0% lethal in female mice. In male mice treated with a 100% lethal dose of picrotoxin, diazepam, 3.0 mg/kg, did not diminish the occurrence of convulsions but reduced the incidence of death to 70%. In equally treated female mice the same dose of diazepam reduced the occurrence of convulsions from 100 to 70% and the incidence of death to 10%. The existence of sex related differences in the response of mice, rats and cats to administration of picrotoxin might have its origin in the dimorphisms of the GABA system in these animal species.     

CCK-antagonist L-364,718: influence on rat pancreatic growth induced by caerulein and bombesin-like peptides

The present study investigates the inhibitory effect of the novel potent benzodiazepine-related CCK-antagonist L-364,718 on pancreatic growth in the rat induced by chronic administration of caerulein and bombesin-like peptides. Caerulein, injected s.c. twice daily at a dose of 1 microgram/kg body weight, and bombesin (10 micrograms/kg) induced a similar increase (1.5-3-fold) in pancreatic wet weight, total protein, amylase, trypsin, putrescine and spermidine content after 14 days of treatment. Growth induced by caerulein showed a significant increase in total DNA content suggesting cellular hyperplasia, whereas bombesin-like peptides led to cellular hypertrophy. In comparison to bombesin the decapeptide neuromedin C (10 micrograms/kg) was found to be 30-50% less potent. In the same dose range, neuromedin B and the tachykinins neurokinin A and B, all structurally related to bombesin, had no significant trophic effect on the rat pancreas. Administration of the CCK-antagonist L-364,718 twice daily at a dose of 0.1 mg/kg or at 1.0 mg/kg, either s.c. or orally, led dose-dependently to a near-complete inhibition of the caerulein-induced trophic effect. In contrast, L-364,718 administered at identical dosages, did not affect pancreatic hypertrophy induced by bombesin and neuromedin C. It is concluded that both peptides mediate their effect on the rat pancreas directly and not via release of endogenous cholecystokinin. Tachykinins are not involved in the regulation of pancreatic growth. Caerulein- and bombesin-like peptides have comparable effects on the stimulation of protein and polyamine synthesis.     

Antiepileptic effects of nifedipine]

In experiments on freely moving male Wistar rats it was shown that nifedipine in a dose 10 mg/kg (i.p.) suppressed the penicillin-induced focal epileptic activity in cerebral cortex. A similar suppressing effect of nifedipine was shown on acute generalized tonic-clonic pentylenetetrazol (PTZ) seizures (75 mg/kg, i.p.). Nifedipine in the same dose was not effective on chronic PTZ administration (PTZ-kindling, 30 mg/kg i.p. during 28 days): when injected 30 min before each PTZ administration it didn't delay the development of kindling induced seizure susceptibility and had no effect on the severity of seizures. The administration of nifedipine in a dose of 10 or 30 mg/kg to control kindled animals which had not been treated with nifedipine had no influence on the severity of seizures provoked by a testing dose of PTZ (30 mg/kg i.p.): its intensity was similar to that of caused by PTZ injection along.     

Nonuniform effect of prolonged haloperidol administration on the GABA(A) and benzodiazepine receptors in different parts of the brain

The experiments on male mice and rats have revealed reversed behavioral effects of muscimol and Ro 15-1788 after 15 days of haloperidol (0.25 mg/kg, twice daily) treatment. Muscimol (0.75 mg/kg), which depressed motor activity in saline-pretreated mice, stimulated it after discontinuation of long-term haloperidol administration. Ro 15-1788 stimulating effect in saline-pretreated rats gave way to sedative effect following haloperidol withdrawal. Simultaneously, the number of 3H-muscimol and 3H-flunitrazepam binding sites was decreased in forebrain, but increased in hindbrain. It was suggested that GABAA and benzodiazepine receptors in forebrain and hindbrain play opposite (inhibiting and stimulating, respectively) functional roles in the regulation of behaviour.     

Ketamine prevents seizures and reverses changes in muscarinic receptor induced by bicuculline in rats

The cholinergic system has been implicated in several experimental epilepsy models. In a previous study bicuculline (BIC), known to antagonize GABA-A postsynaptic receptor subtype, was administered to rats at subconvulsant (1 mg/kg) and convulsant (7.5 mg/kg) doses and quinuclidinyl benzilate ([³H]-QNB) binding to CNS membranes was determined. It was observed that ligand binding to cerebellum increases while it decreases in the case of hippocampus. Saturation binding curves showed that changes were due to the modification of receptor affinity for the ligand without alteration of binding site number. The purpose of this study was to assay muscarinic receptors employing other BIC dose (5 mg/kg), which induces seizures and allows the analysis of a postseizure stage as well. To study further muscarinic receptor involvement in BIC induced seizures, KET was also employed since it is a well known anticonvulsant in some experimental models. The administration of BIC at 5 mg/kg to rats produced a similar pattern of changes in [³H]-QNB binding to those recorded with 1.0 and 7.5 mg/kg doses. Here again, changes were observed in receptor binding affinity without alteration in binding site number for cerebellum or hippocampus membranes. Pretreatment with KET (40 mg/kg) prevented BIC seizures and reverted [³H]-QNB binding changes induced by BIC administration. The single administration of KET invariably resulted in [³H]-QNB binding decrease to either cerebellar or hippocampal membranes. KET added in vitro decreased ligand binding likewise. Results of combined treatment with KET plus BIC are hardly attributable to the single reversion of BIC effect since KET alone invariably decreased ligand binding. It is suggested that besides alteration of cholinergic muscarinic receptor other(s) neurotransmitter system(s) may well also be involved.     

Neurotoxicity of Lindane and Picrotoxin: Neurochemical and Electrophysiological Correlates in the Rat Hippocampus In Vivo

In the present study, we compared in vivo changes of extracellular amino acid levels and nucleotide derivatives to a single ip dose of lindane (10-60 mg/kg) and picrotoxin (5 mg/kg) in the hippocampus of halothane anaesthetized rat by microdialysis-coupled HPLC analysis. Brain activity was monitored by EEG. The effects of lindane and picrotoxin on EEG pattern of rats as well as on hippocampal amino acid and nucleotide status were studied in 0-50 min, 50-100 min and 100-150 min periods post-dosing. Significant decreases in Glu and Asp were found after picrotoxin treatment. After 50-100 min post-dosing, hippocampal hypoxanthine and inosine levels increased to both lindane (10 mg/kg) and picrotoxin whereas xanthine and uridine levels increased to picrotoxin, only. Lindane elicited a dose-dependent occurrence of negative spikes accompanied with rhythmic activity at 4-5 Hz. The picrotoxin-induced 4-5 Hz activity did not display negative sharp waves and was accompanied by 10 Hz oscillations.     

Sex differences in the response of rats to drugs affecting GABAergic transmission

The administration of diazepam 1.0 mg/kg decreased the level of plasma corticosterone in female but not in male Wistar rats. Picrotoxin, another drug affecting GABAergic transmission, also brought about an increase of plasma corticosterone in both sexes. However, in order to achieve a plasma corticosterone increase of similar magnitude (more than 500%) a threefold higher dose of picrotoxin had to be given to males. When the convulsive properties of picrotoxin were tested, it became evident that the dose of picrotoxin (2.5 mg/kg) which was subconvulsive in male was almost 100% convulsive in female rats. The existing sex differences in the response of rats to drugs affecting GABAergic transmission might have possible implications in the treatment of GABA system dysfunction.